stat6 in 3  (MedChemExpress)

Journal: Molecular Therapy. Nucleic Acids

Article Title: STAT6-targeting antisense oligonucleotides against solitary fibrous tumor

doi: 10.1016/j.omtn.2024.102154

Figure Lengend Snippet: Generation of human Lf-ns cells using CRISPR (A) Schematic illustration of the homologous recombination process to create the specific fusion type. (B) Long-range genomic DNA and RNA RT-PCR assays confirmed that Lf-ns cells are heterozygous. (C) Sanger sequencing result of RT-PCR amplicons confirmed the fusion type as NAB2exon6-STAT6exon17. (D) ICC assay showed prominent nuclear expression of NAB2-STAT6 fusion protein. (Red) STAT6 expression by anti-STAT6 antibody conjugated with Alexa Fluor 594, (blue) nuclear staining by DAPI.

Article Snippet: Some high-affinity ASOs have been reported to induce hepatoxicity and immune system activation in vivo , potentially due to their immunostimulatory nature or hybridization-mediated off-target effects., , To probe the in vivo tolerability of our three candidate STAT6 3′ UTR-targeting ASOs (993523, 993562, and 993563), four female C57BL/6 mice per ASO group were treated with intraperitoneal injections of 50 mg/kg ASO twice a week for up to 24 days ( A).

Techniques: CRISPR, Homologous Recombination, Reverse Transcription Polymerase Chain Reaction, Sequencing, Immunocytochemistry, Expressing, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: STAT6-targeting antisense oligonucleotides against solitary fibrous tumor

doi: 10.1016/j.omtn.2024.102154

Figure Lengend Snippet: Engineered and primary SFT cell models that were used in this study

Article Snippet: Some high-affinity ASOs have been reported to induce hepatoxicity and immune system activation in vivo , potentially due to their immunostimulatory nature or hybridization-mediated off-target effects., , To probe the in vivo tolerability of our three candidate STAT6 3′ UTR-targeting ASOs (993523, 993562, and 993563), four female C57BL/6 mice per ASO group were treated with intraperitoneal injections of 50 mg/kg ASO twice a week for up to 24 days ( A).

Techniques: In Vivo, CRISPR, Knock-In, Isolation

Journal: Molecular Therapy. Nucleic Acids

Article Title: STAT6-targeting antisense oligonucleotides against solitary fibrous tumor

doi: 10.1016/j.omtn.2024.102154

Figure Lengend Snippet: In vitro efficacy of unaided delivery of STAT6 3′ UTR-targeting ASOs (A) Schematic illustration of where along exon 22 of STAT6 the 8 STAT6 3′ UTR-targeting ASOs bind. (B) 993523 showed the highest efficacy (62% suppression) against both wild-type (WT) STAT6 and NAB2-STAT6 fusion transcripts in Moffitt-ns cells with gymnotic delivery at concentration of 1 μM after 48 h. (C) In Moffitt-ns cells, 993523 efficiently suppressed both WT STAT6 and NAB2-STAT6 fusion transcripts (IC 50 of 199.4 nM). 993523 efficiently suppressed both the NAB2-STAT6 fusion and WT STAT6 transcripts in (D) Lf-ns cells (IC 50 of 299.9 nM and 206.3 nM). (E) INT-SFT cells (IC 50 of 115.7 nM and 199.5 nM), and (F) IEC139 cells (IC 50 of 250.8 nM and 201.7 nM).

Article Snippet: Some high-affinity ASOs have been reported to induce hepatoxicity and immune system activation in vivo , potentially due to their immunostimulatory nature or hybridization-mediated off-target effects., , To probe the in vivo tolerability of our three candidate STAT6 3′ UTR-targeting ASOs (993523, 993562, and 993563), four female C57BL/6 mice per ASO group were treated with intraperitoneal injections of 50 mg/kg ASO twice a week for up to 24 days ( A).

Techniques: In Vitro, Concentration Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: STAT6-targeting antisense oligonucleotides against solitary fibrous tumor

doi: 10.1016/j.omtn.2024.102154

Figure Lengend Snippet: IC 50 values for STAT6 3′ UTR-targeting ASO inhibition of either NAB2-STAT6 or STAT6 transcript expression in SFT cell models

Article Snippet: Some high-affinity ASOs have been reported to induce hepatoxicity and immune system activation in vivo , potentially due to their immunostimulatory nature or hybridization-mediated off-target effects., , To probe the in vivo tolerability of our three candidate STAT6 3′ UTR-targeting ASOs (993523, 993562, and 993563), four female C57BL/6 mice per ASO group were treated with intraperitoneal injections of 50 mg/kg ASO twice a week for up to 24 days ( A).

Techniques: Inhibition, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: STAT6-targeting antisense oligonucleotides against solitary fibrous tumor

doi: 10.1016/j.omtn.2024.102154

Figure Lengend Snippet: In vitro efficiency of liposome-mediated delivery of STAT6 3′ UTR-targeting ASOs RNAiMAX-mediated delivery of 40 nM of ASOs 993523 or 993562 efficiently decreased the expression of (A) STAT6 and (B) NAB2-STAT6 fusion transcripts in IEC139 cells at both 48 and 72 h. (C) RNAiMAX-mediated delivery of ASO 993523 suppressed IEC139 cell proliferation at both 48 and 72 h. For statistical analysis, two-tailed t-tests were conducted. ∗p < 0.05; ∗∗p < 0.01; n.s., no significant difference.

Article Snippet: Some high-affinity ASOs have been reported to induce hepatoxicity and immune system activation in vivo , potentially due to their immunostimulatory nature or hybridization-mediated off-target effects., , To probe the in vivo tolerability of our three candidate STAT6 3′ UTR-targeting ASOs (993523, 993562, and 993563), four female C57BL/6 mice per ASO group were treated with intraperitoneal injections of 50 mg/kg ASO twice a week for up to 24 days ( A).

Techniques: In Vitro, Expressing, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: STAT6-targeting antisense oligonucleotides against solitary fibrous tumor

doi: 10.1016/j.omtn.2024.102154

Figure Lengend Snippet: Characterization of the IEC139 primary cells and PDX models The NAB2exon6-STAT6exon16 fusion type was confirmed by harvesting the total RNA from (A) IEC139 primary cells and (B) PDX tumors and subsequent RT-PCR and Sanger sequencing. (C) Hematoxylin and eosin staining of PDX tumor tissues showed hypercellular proliferation of fusiform cells, with enlarged “staghorn” blood vessels (black asterisks) and collagenous components (red asterisks). A grade III SFT was indicated by the high number of mitoses (yellow arrowheads), marked atypia, nuclear pleomorphism, and necrotic areas (red arrowheads). (D) IHC assays showed strong nuclear staining for STAT6 (brown) in the PDX tumor tissues. (E) IHC assays showed strong membrane staining for CD34 (brown) in the PDX tumor tissues.

Article Snippet: Some high-affinity ASOs have been reported to induce hepatoxicity and immune system activation in vivo , potentially due to their immunostimulatory nature or hybridization-mediated off-target effects., , To probe the in vivo tolerability of our three candidate STAT6 3′ UTR-targeting ASOs (993523, 993562, and 993563), four female C57BL/6 mice per ASO group were treated with intraperitoneal injections of 50 mg/kg ASO twice a week for up to 24 days ( A).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Staining, Membrane

Journal: Molecular Therapy. Nucleic Acids

Article Title: STAT6-targeting antisense oligonucleotides against solitary fibrous tumor

doi: 10.1016/j.omtn.2024.102154

Figure Lengend Snippet: In vivo tolerability testing of STAT6 3’-UTR-targeting ASOs (A) Schematic illustrating the injection interval and frequency for the in vivo tolerability testing experiments. (B) The animals’ averaged body weight did not significantly change during the treatment course with ASOs 993523, 993562, and 993563 (n = 4). (C) ASOs 993523, 993562, and 993563 did not induce a significant change in the liver, kidney, or spleen of mice (n = 4). (D) ASOs 993562 and 993563 did not induce significant hepatotoxicity in mice (n = 4). ASO 993523 induced a moderate but not clinically precluding increase in serum ALT and AST levels (p < 0.01 using ANOVA with Dunnett’s post hoc tests).

Article Snippet: Some high-affinity ASOs have been reported to induce hepatoxicity and immune system activation in vivo , potentially due to their immunostimulatory nature or hybridization-mediated off-target effects., , To probe the in vivo tolerability of our three candidate STAT6 3′ UTR-targeting ASOs (993523, 993562, and 993563), four female C57BL/6 mice per ASO group were treated with intraperitoneal injections of 50 mg/kg ASO twice a week for up to 24 days ( A).

Techniques: In Vivo, Injection

Journal: Molecular Therapy. Nucleic Acids

Article Title: STAT6-targeting antisense oligonucleotides against solitary fibrous tumor

doi: 10.1016/j.omtn.2024.102154

Figure Lengend Snippet: In vivo efficacy testing of STAT6 3′ UTR-targeting ASOs in IEC139 PDX mouse models (A) Tumor volumes were significantly lower for ASO 993523-treated mice between days 17–24, compared with the untreated control tumors. No significant differences were observed for ASO 993562-treated group (n = 8). (B) Representative tumor samples for each treatment group on day 25. (C) The ASO 993523-treated group showed a significant decrease in tumor weight by day 25 (n = 8). (D) Both ASOs 993523 and 993562 demonstrated significant downregulation of the expression of NAB2-STAT6 fusion transcripts within the tumor tissues by day 25 (n = 8). (E) The ASO 993523-treated group showed a significantly lower mean Ki67 score compared with the control group. (F) Expression levels of NAB2-STAT6 fusion transcripts exhibited a positive correlation with tumor volumes (Pearson correlation coefficient: 0.58; p = 0.003). (G) Expression levels of NAB2-STAT6 fusion transcripts exhibited a positive correlation with Ki67 scores (Pearson correlation coefficient: 0.44; p value: 0.030). For statistical analysis, two-tailed t -tests were conducted. ∗p < 0.05; ∗∗p < 0.01; n.s., no significant difference.

Article Snippet: Some high-affinity ASOs have been reported to induce hepatoxicity and immune system activation in vivo , potentially due to their immunostimulatory nature or hybridization-mediated off-target effects., , To probe the in vivo tolerability of our three candidate STAT6 3′ UTR-targeting ASOs (993523, 993562, and 993563), four female C57BL/6 mice per ASO group were treated with intraperitoneal injections of 50 mg/kg ASO twice a week for up to 24 days ( A).

Techniques: In Vivo, Control, Expressing, Two Tailed Test

Journal: PLoS ONE

Article Title: Omeprazole Blocks STAT6 Binding to the Eotaxin-3 Promoter in Eosinophilic Esophagitis Cells

doi: 10.1371/journal.pone.0050037

Figure Lengend Snippet: mRNA expression levels were determined by quantitative real-time PCR in (A) EoE1-T and (B) EoE2-T cells. Data are the means ± SEM of 3 separate experiments assayed in triplicate. Representative experiments of Western blotting for (C) phospho- and total STAT6 in whole cell lysates and (D) phospho-STAT6 in nuclear lysates in EoE1-T and EoE2-T cells. Tubulin and lamin A/C served as controls for the whole cell and nuclear lysates, respectively. Unstim., unstimulated cells.

Article Snippet: After separation and transfer to nitrocellulose membranes, the membranes were incubated with 1∶1000 dilutions of phospho-STAT6 (Tyr641) or total STAT6 (Cell Signaling), or 1∶2000 dilutions of β-tubulin (Sigma, St. Louis, MO) or 1∶1000 dilutions of lamin A/C (Cell Signaling).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Clinical and Experimental Immunology

Article Title: The effect of short-chain fatty acids on M2 macrophages polarization in vitro and in vivo

doi: 10.1093/cei/uxab028

Figure Lengend Snippet: Acetate, Butyrate, and Propionate inhibit M2 polarization in AMs. MH-S were pretreated with Acetate, Butyrate, and Propionate 30 min at the indicated concentrations before IL-4 (20 ng/ml) exposure for 48 h. ( A, C, E ). Protein expressions of Arg1 and p-Stat6 were evaluated. Β-tubulin was used for normalization. ( B, D, F ) mRNA expressions of M2-associated genes, Arg1, MRC1, Fizz1, and Ym1 were evaluated. Β-actin was used for normalization. Data are shown as means ± SD from three independent experiments. ∗ P < 0.05 vs. CON group. # P < 0.05 vs. IL-4 group.

Article Snippet: All the primary antibodies (Arg1, p-Stat6, β-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), total-Stat6, Cell Signaling Technology, USA; Acetyl-Histone K3, Beyotime Biotechnology, Shanghai; pan Acetyl H3, Merk, Austria) at 1:1000 dilution were incubated over night at 4°C.

Techniques:

Journal: Clinical and Experimental Immunology

Article Title: The effect of short-chain fatty acids on M2 macrophages polarization in vitro and in vivo

doi: 10.1093/cei/uxab028

Figure Lengend Snippet: Acetate, Butyrate, and Propionate inhibit MRC1 and Arg1 protein expression in AMs. MH-S were pretreated with Acetate, Butyrate, and Propionate 30 min at the indicated concentrations before IL-4 (20 ng/ml) exposure for 48 h. ( A ) Percentage of MRC1+ cells were determined by flow cytometry. ( B ) Immunofluorescence staining of the expression of Arg1. Data are shown as means ± SD from three independent experiments. ∗ P < 0.05 vs. CON group. # P < 0.05 vs. IL-4 group.

Article Snippet: All the primary antibodies (Arg1, p-Stat6, β-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), total-Stat6, Cell Signaling Technology, USA; Acetyl-Histone K3, Beyotime Biotechnology, Shanghai; pan Acetyl H3, Merk, Austria) at 1:1000 dilution were incubated over night at 4°C.

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Staining

Journal: Clinical and Experimental Immunology

Article Title: The effect of short-chain fatty acids on M2 macrophages polarization in vitro and in vivo

doi: 10.1093/cei/uxab028

Figure Lengend Snippet: GPR43, but not GPR41 activation may be responsible for the inhibitory effects of Acetate, Butyrate, and Propionate in AMs. MH-S were cultured with IL-4 (20 ng/ml) in the presence or absence of SCFAs (Acetate, Butyrate, and Propionate), GPR41 agonist (AR420626), and GPR43 agonists (4-CMTB) alone or in combination, at the indicated concentrations. ( A ) mRNA expressions of GPR43 and GPR41were evaluated. Β-actin was used for normalization. ( B, C ). Protein expressions of Arg1 and p-Stat6 were evaluated. Β-tubulin was used for normalization. ( D ) mRNA expressions of M2-associated genes, Arg1, MRC1, Fizz1, and Ym1 were evaluated. Β-actin was used for normalization. ( E, F ). Percentage of MRC1+ cells were determined by flow cytometry. Data are shown as means ± SD from three independent experiments. ∗ P < 0.05.

Article Snippet: All the primary antibodies (Arg1, p-Stat6, β-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), total-Stat6, Cell Signaling Technology, USA; Acetyl-Histone K3, Beyotime Biotechnology, Shanghai; pan Acetyl H3, Merk, Austria) at 1:1000 dilution were incubated over night at 4°C.

Techniques: Activation Assay, Cell Culture, Flow Cytometry

Journal: Clinical and Experimental Immunology

Article Title: The effect of short-chain fatty acids on M2 macrophages polarization in vitro and in vivo

doi: 10.1093/cei/uxab028

Figure Lengend Snippet: Butyrate, and Propionate inhibit M2 polarization partly through HDAC inhibition in AMs. MH-S were cultured with IL-4 (20 ng/ml) in the presence or absence of SCFAs (Acetate, Butyrate, and Propionate), HDAC inhibitor (TSA) alone or in combination at the indicated concentrations. ( A ) Representative western blot image and relative expression of H3 acetylation level after 8 h of treatment. Data were normalized against GAPDH. ( B ) Protein expressions of Arg1 and p-Stat6 were evaluated. Β-tubulin was used for normalization. ( C ) mRNA expressions of M2-associated genes, Arg1, MRC1, Fizz1, and Ym1 were evaluated. Β-actin was used for normalization. ( D ) Percentage of MRC1+ cells were determined by flow cytometry. Data are shown as means ± SD from three independent experiments. ∗ P < 0.05 vs. CON group. # P < 0.05 vs. IL-4 group. † P < 0.05 IL-4+Butyrate/Propionate vs. IL-4+Butyrate/Propionate +TSA.

Article Snippet: All the primary antibodies (Arg1, p-Stat6, β-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), total-Stat6, Cell Signaling Technology, USA; Acetyl-Histone K3, Beyotime Biotechnology, Shanghai; pan Acetyl H3, Merk, Austria) at 1:1000 dilution were incubated over night at 4°C.

Techniques: Inhibition, Cell Culture, Western Blot, Expressing, Flow Cytometry

Journal: Clinical and Experimental Immunology

Article Title: The effect of short-chain fatty acids on M2 macrophages polarization in vitro and in vivo

doi: 10.1093/cei/uxab028

Figure Lengend Snippet: Butyrate and Propionate, but not Acetate ameliorate M2 polarization and allergic airway inflammation model. Mice were treated with Acetate, Butyrate, and Propionate in drinking water for 20 days before developing OVA-induced animal model. ( A ) Number of total cells and eosinophils in BALF. ( B ) IL-4, IL-5, and IL-13 levels in lung homogenates of mice. ( C ) Representative images of HE- and PAS-stained histologic sections of the lungs. Scale bars = 100μm. ( D ) Upper: gating strategy. Lower: Percentage of M2 population (MRC1+ cells) in the lung gated from viable CD45+F4/80+CD11c+ cells. ( E ) RT-qPCR was performed to evaluate mRNA levels of Arg1, MRC1, Fizz1, and Ym1 in the lung. Β-actin was used for normalization. Data are shown as means ± SD from three independent experiments. ∗ P < 0.05.

Article Snippet: All the primary antibodies (Arg1, p-Stat6, β-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), total-Stat6, Cell Signaling Technology, USA; Acetyl-Histone K3, Beyotime Biotechnology, Shanghai; pan Acetyl H3, Merk, Austria) at 1:1000 dilution were incubated over night at 4°C.

Techniques: Animal Model, Staining, Quantitative RT-PCR